Published Ahead of Print 16 September 2013. 10.1128/AAC.01016-13.

2013, 57(12):5860. DOI:Antimicrob. Agents Chemother. Edmond J. LaVoie and Daniel S. PilchMalvika Kaul, Lilly Mark, Yongzheng Zhang, Ajit K. Parhi, 

In VivoAntistaphylococcal Efficacy An FtsZ-Targeting Prodrug with Oral

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An FtsZ-Targeting Prodrug with Oral Antistaphylococcal Efficacy InVivo

Malvika Kaul,a Lilly Mark,b Yongzheng Zhang,b Ajit K. Parhi,b Edmond J. LaVoie,c Daniel S. Pilcha

Department of Pharmacology, Rutgers Robert Wood Johnson Medical School, Piscataway, New Jersey, USAa; Taxis Pharmaceuticals, Inc., North Brunswick, New Jersey,USAb; Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers-The State University of New Jersey, Piscataway, New Jersey, USAc

The bacterial cell division protein FtsZ represents a novel antibiotic target that has yet to be exploited clinically. The benzamidePC190723 was among the first FtsZ-targeting compounds to exhibit in vivo efficacy in a murine infection model system. Despiteits initial promise, the poor formulation properties of the compound have limited its potential for clinical development. We de-scribe here the development of an N-Mannich base derivative of PC190723 with enhanced drug-like properties and oral in vivoefficacy. The N-Mannich base derivative (TXY436) is �100-fold more soluble than PC190723 in an acidic aqueous vehicle (10mM citrate, pH 2.6) suitable for oral in vivo administration. At physiological pH (7.4), TXY436 acts as a prodrug, converting toPC190723 with a conversion half-life of 18.2 � 1.6 min. Pharmacokinetic analysis following intravenous administration ofTXY436 into mice yielded elimination half-lives of 0.26 and 0.96 h for the TXY436 prodrug and its PC190723 product, respec-tively. In addition, TXY436 was found to be orally bioavailable and associated with significant extravascular distribution. Usinga mouse model of systemic infection with methicillin-sensitive Staphylococcus aureus or methicillin-resistant S. aureus, we showthat TXY436 is efficacious in vivo upon oral administration. In contrast, the oral administration of PC190723 was not effica-cious. Mammalian cytotoxicity studies of TXY436 using Vero cells revealed an absence of toxicity up to compound concentra-tions at least 64 times greater than those associated with antistaphylococcal activity. These collective properties make TXY436 aworthy candidate for further investigation as a clinically useful agent for the treatment of staphylococcal infections.

Antibiotics in current clinical use are directed at a limited num-ber of targets (e.g., the cell wall, protein synthesis, and DNA

synthesis), many of which are associated with known resistancepathways. As a result, there is a need for new antibiotics with novelmechanisms of action that can help address the growing resistanceproblem. The bacterial protein FtsZ represents a novel drug targetthat has yet to be exploited clinically. FtsZ is a widely conservedbacterial cytoskeletal protein that plays a critical role in cell divi-sion (1). Using GTP as a cofactor, FtsZ self-assembles into poly-mers at the bacterial membrane, forming a Z-ring at the midcellsite of division (1–5). The Z-ring serves as a scaffold for recruit-ment of other key protein constituents of the bacterial divisome(1–4). The essential role that FtsZ plays in cell division makes it anappealing new antibiotic target (6–26).

The substituted benzamide derivative PC190723 (see structurein Fig. 1) was among the first FtsZ-targeting agents to be identified(6, 7). It has been shown to bind FtsZ and stimulate the GTP-dependent polymerization of the protein, which, in turn, leads tothe formation of stable FtsZ polymers that cannot recapitulate thefunction of the Z-ring (13, 27). As a result, bacterial cell division isinhibited, eventually leading to cell death. PC190723 is associatedwith potent and specific activity against Staphylococcus sp., includingmultidrug-resistant strains of methicillin-resistant Staphylococcus au-reus (MRSA) (7, 12). It has also been reported to exhibit in vivo activ-ity in a mouse peritonitis model of S. aureus infection (7).

Although initially promising, the hydrophobic nature ofPC190723 (calculated logP [ClogP] � 2.64) makes it difficult toformulate in vehicles suitable for in vivo treatment, a property thathas limited the potential clinical utility of the compound. We de-scribe here an N-Mannich base derivative of PC190723 (TXY436;see structure in Fig. 1) that is substantially more polar when pro-tonated under acidic conditions (ClogP � �0.51), and formulateseasily in acidic aqueous vehicles suitable for in vivo administra-

tion. We show that TXY436 acts as a prodrug at physiological pH,converting to PC190723. Unlike PC190723, the TXY436 prodrugexhibits antistaphylococcal efficacy in vivo after oral administra-tion in a murine model of systemic infection. In addition, TXY436is minimally toxic to mammalian cells at concentrations up to 64times those associated with antistaphylococcal activity. Theseproperties make TXY436 an attractive lead for future clinical de-velopment.

MATERIALS AND METHODSBacterial strains. S. aureus 8325-4 was a gift from Glenn W. Kaatz (JohnD. Dingell VA Medical Center, Detroit, MI), S. aureus Mu3 was a gift fromGeorge M. Eliopoulos (Beth Israel Deaconess Medical Center, Boston,MA), and Bacillus subtilis FG347 was a gift from Richard Losick (HarvardUniversity, Boston, MA). All other bacterial strains were obtained fromthe American Type Culture Collection (ATCC).

General chemistry methods and synthesis of PC190723 andTXY436. All reactions were done under nitrogen atmosphere. Reactionmonitoring and follow-up were done using aluminum backed Silica GTLC plates with UV254 (Sorbent Technologies), visualizing with UVlight. Flash column chromatography was done on a Combi Flash Rf Tele-dyne ISCO. The 1H (300 MHz) and 13C (75 MHz) nuclear magneticresonance (NMR) spectra were acquired on a Varian Unity Inova (300MHz) multinuclear NMR spectrometer. The data are expressed in partsper million relative to the residual nondeuterated solvent signals, spin

Received 13 May 2013 Returned for modification 20 June 2013Accepted 6 September 2013

Published ahead of print 16 September 2013

Address correspondence to Daniel S. Pilch, pilchds@rwjms.rutgers.edu.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AAC.01016-13

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multiplicities are given as s (singlet), d (doublet), m (multiplet), and bs(broad singlet), and coupling constants (J) are reported in Hertz (Hz).Melting points were determined using a Mel-Temp II apparatus (Labora-tory Devices, Inc.) and are uncorrected.

PC190723 was synthesized as previously described (28). TXY436 wassynthesized as follows. In a sealed tube, a mixture of PC190723 (200 mg,0.56 mmol), formaldehyde (37%, 0.3 ml, 3.7 mmol), and dimethylamine(2 M in tetrahydrofuran [THF], 1.9 ml, 3.70 mmol) in H2O/THF (8/4 ml)was heated at 65°C for 2 h. After cooling to room temperature, the reac-tion mixture was diluted with ethyl acetate, washed with brine, dried overNa2SO4, and concentrated to afford a brown solid. Subsequent purifica-tion by column chromatography afforded the pure product TXY436 as alight brown solid (154 mg, 67% yield): melting point, 152 to 154°C; 1HNMR (CDCl3, 300 MHz); �: 8.57 (d, J � 2.1 Hz, 1H), 8.24 (d, J � 2.1 Hz,1H), 7.17 to 7.09 (m, 1H), 6.92-6.86 (m, 1H), 6.20 (bs, 1H), 5.48 (s, 2H),4.30 (d, J � 6.0 Hz, 2H), 2.37 (s, 6H). HRMS calculated forC17H15ClF2N4O2S (M � H)� 413.0645. Found 413.0656.

Comparator antibiotics and S. aureus FtsZ. Vancomycin-HCl,erythromycin, and oxacillin (sodium salt) were obtained from Sigma-Aldrich Co. S. aureus FtsZ was expressed in Escherichia coli and purified asdescribed previously (21).

Compound solubility studies. The maximum solubilities of PC190723and TXY436 in 10 mM citrate (pH 2.6) or phosphate-buffered saline(PBS; pH 7.4) were determined at 25°C. In these assays, the compoundswere combined with vehicle at weight/volume (wt/vol) ratios above theirsolubility limit, and the amount of dissolved compound quantified. Thewt/vol ratios targeted in the PBS vehicle were 0.2 mg/ml for both com-pounds, while the wt/vol ratios targeted in the citrate vehicle were 0.2mg/ml for PC190723 and 2.5 mg/ml for TXY436. Each solubility assess-ment was conducted in duplicate. All samples were vortexed for 3 min toafford maximal compound dissolution and then centrifuged at 16,000 �g for 1 min to pellet out any unsolubilized compound. The concentrationof compound in each of the resulting supernatants was then quantified byusing reverse-phase high-performance liquid chromatography (HPLC) asdescribed below and generating standard curves of peak area versus com-pound concentration (in citrate or PBS vehicle).

Compound stability studies. Experimental solutions of TXY436 orPC190723 at concentrations of either 10 or 20 �g/ml were prepared from4-mg/ml dimethyl sulfoxide (DMSO) stock solutions in either 10 mMcitrate (pH 2.6) or PBS (pH 7.4) vehicle. The relative amount of TXY436and PC190723 in each experimental solution was then assessed as a func-tion of time at 25°C using reverse-phase HPLC as described below.

Reverse-phase HPLC. For all HPLC measurements, a reverse-phaseSPHER-100 C18 column (Princeton Chromatography, Inc.) was used on aShimadzu LC-20AT liquid chromatograph equipped with a ShimadzuSPD-20AV UV/VIS detector (set at 296 nm). The column size was 150mm by 4.6 mm, with particle and pore sizes of 5 �m and 100 Å, respec-tively. A 20-�l sample of each experimental solution was injected, and aflow rate of 1 ml/min was applied, along with a gradient of 10 to 90%acetonitrile (containing 0.1% [vol/vol] trifluoroacetic acid) and water inthe mobile phase. The total run time was 20 min, with the sampling fre-quency and response time being 2 Hz and 1 s, respectively. Under theseconditions, TXY436 eluted �9.8 min after sample injection, andPC190723 eluted �11.7 min after sample injection. Peak areas were de-termined using the Shimadzu EZStart 7.4 SP3 software package.

MIC assays. MIC assays were conducted in accordance with Clinicaland Laboratory Standards Institute (CLSI) guidelines for broth microdi-lution (29). Briefly, log-phase bacteria were added to 96-well microtiterplates (at 5 � 105 CFU/ml) containing 2-fold serial dilutions of com-pound or comparator drug in cation-adjusted Mueller-Hinton (CAMH)broth, with each compound concentration being present in duplicate. Thefinal volume in each well was 0.1 ml, and the microtiter plates were incu-bated aerobically for 24 h at 37°C. Bacterial growth was then monitored bymeasuring the optical density at 600 nm using a VersaMax plate reader(Molecular Devices, Inc.), with the MIC being defined as the lowest com-pound concentration at which growth was �90% inhibited. The follow-ing S. aureus strains were included in these assays: 8325-4 (methicillin-susceptible S. aureus [MSSA]), ATCC 49951 (mucoid MSSA), ATCC19636 (MSSA), ATCC 29213 (MSSA), ATCC 33591 (MRSA), ATCC43300 (MRSA), and Mu3 (MRSA). As recommended by CLSI (29), theCAMH broth was supplemented with 2% NaCl in all of the MRSA exper-iments so as to maintain selection pressure for MRSA. When present,filtered mouse serum (Lampire Biological Laboratories, Inc.) was used at50% (vol/vol).

MBC assays. Minimum bactericidal concentration (MBC) assayswere conducted in accordance with CLSI guidelines (29). Broth microdi-lution assays were conducted as described in the preceding section. Afterthe 24-h incubation period, aliquots from the microtiter wells were platedonto tryptic soy agar (TSA). The colonies that grew after 24 h of incuba-tion were counted using an Acolyte colony counter (Synbiosis, Inc.), withMBC being defined as the lowest compound concentration resulting in a�3-log reduction in the number of CFU.

Time-kill assays. Exponentially growing S. aureus 8325-4 bacteriawere diluted in CAMH broth to a final count of 105 to 106 CFU/ml. Thecolony count at time zero was verified by plating serial dilutions of theculture in duplicate on TSA plates. The initial culture was aliquoted intotubes, each containing either a compound or comparator drug at finalconcentrations ranging from 0� to 8� the MIC. An equivalent volume ofDMSO was added to the vehicle control tube. The cultures were thenincubated at 37°C with shaking. The CFU/ml in each culture was deter-mined over time by withdrawing samples at time points raging from 2 to24 h and plating appropriate serial dilutions on to TSA plates. To avoidthe possibility of carryover effects in instances when no dilutions weremade of the cultures, the samples were centrifuged at 16,000 � g, thesupernatant was removed, and the bacterial pellet was resuspended incompound-free medium before plating. All TSA plates were incubatedat 37°C, and the CFU/ml at each time point determined by countingcolonies after 24 h.

Phase-contrast microscopy. Log-phase S. aureus 8325-4 cells werecultured in CAMH broth at 37°C for 4 h in the presence of DMSO (solventcontrol), PC190723 at 3 �g/ml, or TXY436 at 3 �g/ml. A 1-ml sample waswithdrawn from each bacterial culture, and centrifuged at 16,000 � g for3 min at room temperature. The supernatant was removed, and the bac-terial pellet was washed with 1 ml of PBS. The final bacterial pellet wasthen resuspended in 50 �l of PBS, with 5 �l of the resulting bacterialsuspension being transferred onto a microscope slide, together with 5 �lof 1% molten agarose (made in PBS). A coverslip was applied, and the

FIG 1 Chemical structures of PC190723 and TXY436. TXY436 is depictedwith the Mannich base functionality in its protonated (cationic) form. Theindicated ClogP values were calculated using the weighted method (VG �KLOP � PHYS � 1) in the Marvin 5.12 Software Suite (ChemAxon, Ltd.),with Cl� and Na�/K� concentrations being set at 0.1 mol/dm3.

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slide was visualized with a Zeiss Axioplan 2 microscope equipped with aPlan-Apochromat �100 objective lens (NA � 1.40). Images were cap-tured with a Zeiss Axiocam HR camera by using the OpenLab softwarepackage.

Fluorescence microscopy. The impact of TXY436 and PC190723 onFtsZ Z-ring formation in B. subtilis FG347 bacteria was assessed in a man-ner similar to that described previously (30). Specifically, exponentiallygrowing FG347 bacteria were cultured in CAMH broth for 1 h at 37°C inthe presence of DMSO (solvent control), PC190723 at 3 �g/ml, orTXY436 at 3 �g/ml. The expression of green fluorescent protein (GFP)-conjugated ZapA was then induced by the addition of xylose to a finalconcentration of 0.25% (wt/vol), and the cells were incubated for 1 addi-tional hour at 37°C. The bacterial cultures were then treated and visual-ized as described above, with the additional incorporation of a standardGFP filter set.

Frequency of resistance assay. The frequency of resistance (FOR) toTXY436 was assayed using a large inoculum approach. In this approach,TSA plates containing TXY436 at concentrations ranging from 4� to 32�the MIC were prepared. A large inoculum of �3 � 109 CFU/ml of S.aureus 8325-4 was spread onto each plate. The colony count of the inoc-ulum was verified by plating serial dilutions of the culture onto nonselec-tive TSA plates. All plates were incubated at 37°C overnight and examinedafter 24 h. Mutational frequency was calculated from the ratio of thenumber of colonies observed on the selective plates to the total number ofplated bacteria (31).

FtsZ polymerization assay. Polymerization of S. aureus FtsZ wasmonitored using a microtiter plate-based spectrophotometric assay inwhich changes in FtsZ polymerization are reflected by correspondingchanges in absorbance at 340 nm (A340). PC190723 or TXY436 (at con-centrations ranging from 0 to 10 �g/ml) were combined with 5 �M FtsZin 100 �l of reaction solution. Reaction solutions contained 50 mM Tris-HCl (pH 7.4), 50 mM KCl, and 10 mM magnesium acetate. Reactionswere assembled in half-volume, flat-bottom, 96-well microtiter plates andinitiated by the addition of 4 mM GTP. Polymerization was continuouslymonitored at 25°C by measuring A340 in a VersaMax plate reader over atime period of 60 min.

Pharmacokinetic studies. Pharmacokinetic experiments were con-ducted by SAI Life Sciences, Ltd. (Pune, India) in accordance with guide-lines established by the Indian Committee for the Purpose of Control andSupervision of Experiments on Animals (CPCSEA). Approval by the In-stitutional Animal Ethics Committee (IAEC) was obtained prior to initi-ation of the studies. Healthy male BALB/c mice (8 to 12 weeks old andweighing between 25 and 35 g) were obtained from ACTREC (Mumbai,India). Twelve mice were divided into two groups of six mice each, withthree mice being housed per cage. Food and water were provided to themice ad libitum. The first group of mice received a single intravenous (i.v.)dose of TXY436 at 5 mg/kg by tail vein injection. The second group re-ceived single peroral (p.o.) dose of TXY436 at 10 mg/kg by gavage. In bothcases, the compound was formulated in 10 mM citrate vehicle (pH 2.6) ata concentration of 0.85 mg/ml. Blood samples (�60 �l) were collectedfrom the retro-orbital plexus at 0.08, 0.25, 0.5, 1, 4, and 8 h after the i.v.dose and 0.25, 0.5, 1, 4, and 8 h after the p.o. dose. The blood samples werecollected from a set of three mice at each time point and placed in micro-centrifuge tubes containing a 20% K2EDTA solution as an anticoagulant.The blood samples were then centrifuged at 4,000 rpm for 10 min at 4°C toseparate the plasma. The plasma samples were stored at �80°C prior totheir bioanalysis. Plasma concentrations of TXY436 and PC190723 werequantified by LC-MS/MS, with the lower limit of quantitation (LLOQ)being 20.2 ng/ml for both compounds. The time-dependent plasma con-centration data were analyzed by using the sparse sampling mode in thenoncompartmental analysis (NCA) tool of the Phoenix WinNonlin ver-sion 6.3 software package to yield relevant pharmacokinetic parameters.

In vivo efficacy studies. Antistaphylococcal efficacy in vivo was as-sessed in a mouse peritonitis model of systemic infection with S. aureusATCC 19636 (MSSA) or ATCC 43300 (MRSA). These studies were con-

ducted in full compliance with the standards established by the U.S. Na-tional Research Council’s Guide for the Care and Use of Laboratory An-imals and were approved by the Institutional Animal Care and UseCommittee of Rutgers University. Groups of six female Swiss-Webstermice with an average weight of 25 g were infected intraperitoneally with alethal inoculum of either MSSA (500 �l of saline containing 8 � 106

CFU/ml and 1.5% [wt/vol] porcine mucin [Sigma-Aldrich Co.]) orMRSA (500 �l of saline containing 108 CFU/ml and 5% [wt/vol] porcinemucin).

The mice were fasted overnight prior to their use in the studies. Allcompound and vehicle administrations were performed p.o. by gavage,with the vehicle being 10 mM citrate (pH 2.6). Both TXY436 andPC190723 were formulated at 2.0 mg/ml, with the resulting TXY436 for-mulation being a solution and the resulting PC190723 formulation beinga suspension.

Four experimental groups of MSSA-infected mice were treated as fol-lows: group 1, untreated; group 2, vehicle only; group 3, TXY436 at 128mg/kg (in four divided doses of 32 mg/kg); and group 4, PC190723 at 128mg/kg (in four divided doses 32 mg/kg). The first dose of compound wasadministered 10 min after infection, with subsequent doses being admin-istered at 12-min intervals thereafter. In the MRSA studies, three experi-mental groups of infected mice were treated as follows: group 1, untreat-ed; group 2, vehicle only; and group 3, TXY436 at 192 mg/kg (in sixdivided doses of 32 mg/kg). In these studies, the first dose of compoundwas administered 1 h after infection, with subsequent doses being admin-istered at 12-min intervals thereafter. The dosing volume for all of the p.o.administrations was 16 ml/kg.

An additional group of six infected mice was also included as a positivecontrol group in both the MSSA and the MRSA studies. This group wastreated with a single i.v. administration of vancomycin (formulated insaline) at a dose of 16 mg/kg in the MSSA studies and 24 mg/kg in theMRSA studies. The vancomycin was administered 10 min after MSSAinfection, while being administered 1 h after MRSA infection. The dosingvolume for the i.v. vancomycin control groups was 8 ml/kg.

The body temperatures of all mice were monitored for a period of 5days after infection. Body temperatures were recorded at the Xiphoidprocess using a noninvasive infrared thermometer (Braintree Scientific,Inc.). Infected mice with body temperatures of �28.9°C were viewed asbeing unable to recover from the infection (32) and were euthanized.

MTT cytotoxicity assay. The cytotoxicity of TXY436 versus Vero cells(African green monkey kidney epithelial cells; ATCC) was assessed usinga 72-h continuous 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay as described previously (33).

RESULTS AND DISCUSSIONTXY436 is associated with a 100-fold enhanced solubility rela-tive to PC190723 in an acidic aqueous vehicle (10 mM citrate,pH 2.6) suitable for in vivo drug administration. We sought todetermine whether the N-Mannich base functionality on TXY436afforded the compound enhanced aqueous solubility relativeto PC190723. To this end, we used reverse-phase HPLC todetermine the maximal solubilities of TXY436 and PC190723in two different aqueous vehicles with differing values of pH:10 mM citrate (pH 2.6) and PBS (pH 7.4). The maximal solu-bilities derived from these studies are listed in Table 1. Thesolubility of TXY436 is �2.8-fold greater than that ofPC190723 in PBS at pH 7.4 (66.8 15.7 �g/ml for TXY436versus 23.9 4.0 �g/ml for PC190723), while being �100-foldgreater in citrate at pH 2.6 (2,290 199 �g/ml for TXY436versus 22.5 1.6 �g/ml for PC190723). Thus, the N-Mannichbase functionality affords TXY436 increased aqueous solubilityrelative to PC190723, with the degree of solubility enhance-ment being significantly greater at acidic pH (2.6) than at phys-iological pH (7.4). Importantly, the solubility of TXY436 in the

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acidic citrate vehicle (2,290 �g/ml) is sufficient in magnitudefor in vivo efficacy studies of TXY436 using this vehicle.

TXY436 is chemically stable in the acidic citrate vehicle. Wenext sought to assess the chemical stability of TXY436 in the acidiccitrate vehicle over time using reverse-phase HPLC. Figure 2shows the HPLC chromatogram of TXY436 at 10 �g/ml after 24 hof incubation in the acidic citrate vehicle at 25°C, with the corre-sponding chromatogram of PC190723 at 10 �g/ml also shown forcomparative purposes. Under the conditions used in these HPLCmeasurements (detailed in Materials and Methods), TXY436elutes at �9.8 min after injection, while PC190723 elutes at �11.7min. The shorter retention time of TXY436 relative to PC190723is consistent with its correspondingly decreased hydrophobicity(ClogP � �0.51 for TXY436 and �2.64 for PC190723). Note thatthe chromatographic profile of TXY436 remained essentially un-changed, even after 72 h of incubation (data not shown), indicat-ing that TXY436 is chemically stable over this period of time in theacidic citrate vehicle.

Although stable at pH 2.6, TXY436 converts to PC190723 atpH 7.4 with a half-life (t1/2) of 18.2 � 1.6 min at 25°C. N-Man-nich base derivatives of drugs are susceptible to pH-dependentchemical hydrolysis, which has been used as a platform for makingprodrugs with improved formulation and delivery properties (34,35). Rolitetracyclin (transcycline) is an example of a clinically usedN-Mannich base prodrug of an antibiotic (tetracycline) (35). Oneof the by-products of N-Mannich base hydrolysis is formaldehyde(34, 35). Although the release of formaldehyde after N-Mannichbase prodrug hydrolysis raised concerns early on regarding poten-tial toxicity, these concerns have been allayed by studies demon-

strating that the level of formaldehyde exposure is well below thetoxic threshold (36).

If TXY436 were susceptible to pH-dependent chemical hydro-lysis like other N-Mannich base derivatives, one of the expectedproducts would be PC190723. We used reverse-phase HPLC toprobe for the potential conversion of TXY436 to PC190723 insolution at the physiological pH of 7.4. To this end, we monitoredthe chemical stability of TXY436 over time in PBS (pH 7.4) at25°C. Figure 3A shows the HPLC chromatograms of TXY436 at 20�g/ml after incubation in PBS for various times over a period of 60

TABLE 1 Solubilities of TXY436 and PC190723 in 10 mM citrate andPBS at 25°Ca

Compound

Mean solubility (�g/ml) SD

Citrate (pH 2.6) PBS (pH 7.4)

TXY436 2,290 199 66.8 15.7PC190723 22.5 1.6 23.9 4.0a Each solubility value reflects the average of two independent determinations.

FIG 2 Reverse-phase HPLC chromatograms of TXY436 and PC190723 afterincubation for 24 h in 10 mM citrate (pH 2.6) at 25°C. The concentration ofeach compound was 10 �g/ml.

FIG 3 (A) Reverse-phase HPLC chromatograms of TXY436 at 20 �g/ml afterthe indicated times of incubation in PBS (pH 7.4) at 25°C. For comparativepurposes, the corresponding chromatogram of PC190723 at 20 �g/ml after 60min of incubation in PBS is also presented. The solid arrow indicates the peakcorresponding to TXY436, while the dashed arrow indicates the peak corre-sponding to PC190723. (B) Plot of ln(peak area) versus time for the peakcorresponding to TXY436. The indicated half-life (t1/2) for the conversion ofTXY436 to PC190723 was determined using the relationship, t1/2 � �0.693/m,where m is the slope derived from the linear least-squares fit of the experimen-tal data points (as indicated by the solid line). The uncertainty in t1/2 reflectsthe maximal error in m propagated through the above relationship.

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min. The chromatogram of PC190723 at 20 �g/ml after 60 min ofincubation in PBS is also shown as a reference. Note that over thistime period, TXY436 becomes almost fully converted toPC190723. Thus, in essence, TXY436 acts as a stable acid-solubleprodrug that converts to PC190723 under physiological pH condi-tions. A linear regression analysis of the time-dependent decrease inthe peak areas of TXY436 indicates that the TXY436-to-PC109723conversion follows first-order kinetics, while also yielding a half-life(t1/2) for the conversion of 18.2 1.6 min (Fig. 3B).

The TXY436 prodrug retains the antistaphylococcal andFtsZ-targeting activities of the PC190723 product. For an N-Mannich base prodrug such as TXY436 to be active in vivo, it mustmaintain the same biological activities in vitro as the product(PC190723) itself. We therefore compared TXY436 andPC190723 with regard to antistaphylococcal activity (as indicatedby MIC, MBC, and kinetics of kill), as well as FtsZ-targeting ac-tivity (as reflected by the impact on bacterial morphology, FtsZpolymerization, and Z-ring formation).

Antistaphylococcal activity of TXY436. Our initial studiescompared the activities of TXY436 and PC190723 against fourdifferent MSSA and three different MRSA strains, with the result-ing MICs being summarized in Table 2. The MICs of TXY436 weresimilar, if not identical, to those of PC190723 against all of theMSSA and MRSA strains examined, with these MICs rangingfrom 0.5 to 1.0 �g/ml. With regard to the three MRSA strains usedin the studies, the activities of TXY436 were comparable to theactivity of the prototypical anti-MRSA drug vancomycin (MIC �2.0 �g/ml), which was included as a comparator antibiotic. Oxa-cillin (also included as a control agent) was predictably inactiveversus the MRSA strains (MIC 64 �g/ml).

We next evaluated the antistaphylococcal activities of TXY436and PC190723 with regard to their bactericidal behavior. As a firststep toward this goal, we determined the MBC values of TXY436and PC190723 against two of the MSSA strains (8325-4 and ATCC19636) and two of the MRSA strains (ATCC 33591 and 43300).The bactericidal antistaphylococcal drug vancomycin and thebacteriostatic drug erythromycin were used as comparator con-trols in these determinations. In accordance with CLSI standards,an MBC/MIC ratio of 1 to 2 is considered indicative of bactericidalbehavior (29). In contrast, an MBC/MIC ratio �8 is viewed asbeing indicative of bacteriostatic behavior. As expected, the con-trol bactericidal agent vancomycin yielded MBC/MIC ratios of 1to 2 against all of the MSSA and MRSA strains examined (Table 3).Also, as expected, the control bacteriostatic drug erythromycinyielded MBC/MIC ratios of 128 against the two MSSA strains.

Both MRSA strains were resistant to erythromycin, precludingMBC determinations against these strains. Significantly, TXY436and PC190723 exhibited MBC/MIC ratios of 1 to 2 against all ofthe MSSA and MRSA strains, indicative of bactericidal antistaphy-lococcal behavior.

In addition to assessing the bactericidal activities of TXY436and PC190723 against S. aureus with regard to MBC/MIC ratios,we also probed for bactericidal activity using a time-kill assay atcompound concentrations of 1�, 2�, 4�, and 8� MIC. Figure 4shows representative time-kill curves, with vancomycin being in-cluded as a comparator control agent. Inspection of these time-killdata reveals that both TXY436 and PC109723 produce 3 logs ofkill at concentrations of 2� the MIC or greater. Significantly, thisobservation is consistent with the bactericidal behavior revealedby our MBC/MIC analysis described above. It is also of interest tonote that at concentrations of �2� the MIC, the killing kinetics ofTXY436 and PC190723 are more rapid than those of vancomycin.

PC190723 has been shown to be associated with a frequency ofresistance (FOR) in S. aureus of approximately 3 � 10�8 (7, 37).We sought to determine the corresponding FOR to the TXY436prodrug in S. aureus using the large inoculum approach describedin Materials and Methods. These studies yielded an FOR toTXY436 of (2.0 0.7) � 10�8, in agreement with the previouslyreported value for PC190723.

FtsZ-targeting activity of TXY436. One of the hallmarks ofFtsZ-targeting compounds that disrupt bacterial cell division isthe induction of an enlarged morphology in treated bacteria (7,12, 27, 37). Therefore, in our initial studies probing the FtsZ-targeting activity of TXY436, we determined the impact of thecompound on the morphology of S. aureus bacteria using phase-contrast microscopy. Control DMSO-treated S. aureus bacteriaare �1 �m in diameter, in agreement with previously reported

TABLE 2 Antibacterial activities of TXY436 and PC190723 againstMSSA and MRSAa

Strain

MIC (�g/ml)

TXY436 PC190723 Vancomycin Oxacillin

8325-4 (MSSA) 0.5 0.5 1.0 0.125ATCC 29213 (MSSA) 1.0 1.0 1.0 0.25ATCC 19636 (MSSA) 0.5 0.5 1.0 0.125ATCC 49951 (MSSA) 1.0 0.5 1.0 0.25ATCC 33591 (MRSA) 0.5 0.5 2.0 64ATCC 43300 (MRSA) 0.5 0.5 2.0 64Mu3 (MRSA) 0.5 0.5 2.0 64a Vancomycin and oxacillin are included as control antistaphylococcal agents. Mu3 is aclinical MRSA isolate also identified as being hetero-GISA (hGISA) (43).

TABLE 3 Comparison of MICs and MBCs for TXY436 and PC190723against MSSA and MRSAa

Strain and compound MIC (�g/ml) MBC (�g/ml) MBC/MIC

8325-4 (MSSA)TXY436 0.5 1.0 2PC190723 0.5 1.0 2Vancomycin 1.0 2.0 2Erythromycin 0.125 32 256

ATCC 19636 (MSSA)TXY436 0.5 0.5 1PC190723 0.5 0.5 1Vancomycin 1.0 1.0 1Erythromycin 0.5 64 128

ATCC 33591 (MRSA)TXY436 0.5 1.0 2PC190723 0.5 1.0 2Vancomycin 2.0 4.0 2Erythromycin 64 NA NA

ATCC 43300 (MRSA)TXY436 0.5 1.0 2PC190723 0.5 1.0 2Vancomycin 2.0 4.0 2Erythromycin 64 NA NA

a Vancomycin and erythromycin are included as control bactericidal and bacteriostaticagents, respectively. NA, not applicable.

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diameters characteristic of S. aureus (38) (Fig. 5A). In contrast, theTXY436-treated bacteria exhibited 2- to 3-fold-enlarged pheno-types, with diameters ranging from 2 to 3 �m (Fig. 5B).PC190723-treated bacteria exhibited a similar behavior (Fig. 5C).Thus, TXY436 is able to induce the type of morphological changein S. aureus that is a hallmark of FtsZ-directed inhibitors of celldivision such as PC190723 (6, 7).

Stimulation of FtsZ polymerization dynamics and stabilizationof nonfunctional FtsZ polymeric structures are thought to under-lie the antibacterial activities of FtsZ-targeting agents such asPC190723 (7, 13, 27, 37, 39). We probed for the propensity ofTXY436 to exert these types of effects on the polymerization of

purified S. aureus FtsZ in vitro. For this assay, we used a microtiterplate-based spectrophotometric approach in which FtsZ poly-merization is detected in solution by a time-dependent increase insolution absorbance at 340 nm (A340). Figure 6A shows the time-dependent A340 profiles of S. aureus FtsZ in the absence or pres-ence of TXY436 at concentrations ranging from 1 to 10 �g/ml.The corresponding results for PC190723 are also included forcomparative purposes (Fig. 6B). Note that TXY436 stimulatesFtsZ polymerization to a similar extent as PC190723, with themagnitudes of these stimulatory effects also being similarly de-pendent on compound concentration. Vancomycin was includedas a non-FtsZ-targeting control drug in these assays. As expected,vancomycin did not impact the polymerization of S. aureus FtsZto any significant degree.

In addition to investigating the impact of TXY436 on the func-tion of purified FtsZ in vitro, we also sought to examine the impactof the compound on FtsZ function in live bacteria. To this end, wemonitored the impact of the compound on the formation of FtsZZ-rings using a strain of B. subtilis (FG347) that inducibly ex-presses GFP-tagged ZapA, a known protein marker for FtsZ Z-rings (30). We treated the FG347 bacteria with DMSO (solventcontrol), TXY436, or PC190723 and examined the bacteria usingfluorescence microscopy. In the absence of compound, fluores-cent foci corresponding to Z-rings are evident at midcell (high-lighted by the arrows in Fig. 7A). In contrast, the TXY436-treatedbacteria lack these midcell foci, instead exhibiting punctate fluo-rescent foci distributed throughout each cell (Fig. 7B). Recall thatTXY436 acts as an enhancer of FtsZ polymerization. It is thereforelikely that the punctate fluorescent foci distributed throughoutthe TXY436-treated bacteria reflect multiple nonfunctional FtsZpolymeric structures distinct from Z-rings. This type of behavioris characteristic of agents such as PC190723 that stabilize FtsZpolymers (Fig. 7C) (6, 7). In addition, the TXY436-treated bacte-ria, like the PC190723-treated bacteria, are significantly moreelongated (filamentous) relative to the control DMSO-treatedbacteria, a morphological change typical of rod-shaped bacteria inwhich cell division has been inhibited through disruption of FtsZfunction (6, 7, 30, 40–42).

Taken together, the MIC/MBC, time-kill, microscopy, and FtsZpolymerization results described in the preceding sections indicatethat the TXY436 prodrug maintains all the antistaphylococcal andFtsZ-targeting activities of the PC190723 product itself.

TXY436 retains antistaphylococcal activity in the presence ofmouse serum. An important criterion for a compound with re-gard to its potential for exhibiting antibacterial efficacy in vivo isfor it to retain antibacterial activity in the presence of serum. Inrecognition of this important property, we assessed the impact of50% (vol/vol) mouse serum on the activity of TXY436 against S.aureus. As highlighted in Table 4, the presence of mouse serumresulted in a 4-fold reduction in the antistaphylococcal potency ofTXY436 (with the MIC increasing from 0.5 �g/ml in the absence ofserum to 2.0 �g/ml in the presence of serum). A similar reduction inpotency was observed for PC190723 itself. Note that the MIC ofTXY436 in the presence of serum is identical to the correspondingMIC for the control drug vancomycin. Thus, TXY436 is still able toretain antistaphylococcal activity in the presence of serum.

Pharmacokinetic analysis of the TXY436 prodrug and itsPC190723 product after both intravenous and oral administra-tion of TXY436 to mice reveals the prodrug product to be 73%orally bioavailable. As a prelude to conducting in vivo efficacy

FIG 4 Time-kill curves for S. aureus 8325-4 (MSSA). Each data point reflectsthe average of two independent measurements, with the error bars reflectingthe standard deviation from the mean. Bacteria were treated with DMSO ve-hicle only (no drug), TXY436 (A), PC190723 (B), or vancomycin (C). Allagents were used at concentrations ranging from 1� to 8� the MIC.

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experiments, we sought to evaluate the plasma pharmacokineticsof the TXY436 prodrug and its PC190723 product after both i.v.and p.o. administration of TXY436 to mice. In these studies, asingle i.v. dose of 5 mg/kg or p.o. dose of 10 mg/kg was adminis-tered to mice, and the plasma concentrations of the TXY436 pro-drug and the PC190723 product were measured at time pointsranging from 0.08 to 8 h using LC-MS. For both the i.v. and thep.o. studies, 10 mM citrate (pH 2.6) was used as the vehicle. Theplasma concentration of TXY436 was detectable and quantifiableup to 1 h after i.v. administration and up to 2 h after p.o. admin-istration (Fig. 8A). The plasma concentration of PC190723 wasdetectable and quantifiable up to 4 h after i.v. administration andup to 8 h after p.o. administration (Fig. 8B).

Pharmacokinetic analyses of the time-dependent plasma con-centration profiles for TXY436 and PC190723 with the NCA mod-ule of the Phoenix WinNonlin version 6.3 software packageyielded the pharmacokinetic parameters listed in Table 5. Analysisof the TXY436 plasma concentrations after i.v. administrationyielded an elimination half-life (t1/2) of 0.26 h and a high totalbody clearance (CL) of 312 ml/min·kg, �3.5-fold that of normalliver blood flow in mice (90 ml/min·kg). The volume of distribu-tion at steady state (Vss) was found to be 5.77 liters/kg. Signifi-cantly, this value of Vss is �8.2-fold greater than that of normalbody water (0.7 liters/kg), indicating substantive extravasculardistribution of the compound. After p.o. administration ofTXY436, the peak plasma concentration was achieved within 30min (tmax � 0.5 h). A comparison of the areas under the curves upto the last detectable concentrations of TXY436 (AUClast) afterboth p.o. and i.v. administration yields an absolute oral bioavail-ability (%F) for the prodrug itself of 33% after normalization forthe dose.

Analysis of the PC190723 plasma concentrations after i.v. ad-ministration of TXY436 yielded an �4-fold-longer t1/2 (0.96 h)than that of TXY436 and an �3.6-fold-lower CL of 86.0 ml/min-kg, a value that is comparable to normal liver blood flow. The Vss

of PC190723 (5.39 liters/kg) was similar to that of TXY436, indic-ative a similar degree of extravascular distribution. After p.o. ad-ministration of TXY436, the peak plasma concentration ofPC190723 was achieved within 30 min (tmax � 0.5 h). A compar-ison of the AUClast values of PC190723 after both p.o. and i.v.administration of TXY436 yields an absolute oral bioavailabilityfor the prodrug product of 73% after normalization for dose.

FIG 5 Phase-contrast micrographs of S. aureus 8325-4 bacteria cultured for 4 h in the presence of DMSO (solvent control) (A), TXY436 at 3 �g/ml (B), orPC190723 at 3 �g/ml (C).

FIG 6 Concentration dependence of the impact of TXY436 (A) or PC190723(B) on the polymerization of S. aureus FtsZ, as determined by monitoringtime-dependent changes in absorbance at 340 nm (A340) at 25°C. The time-dependent A340 polymerization profiles were acquired in the presence ofDMSO (solvent control) or the indicated concentrations of compound (rang-ing from 1 to 10 �g/ml). The polymerization profile acquired in the presenceof vancomycin at 10 �g/ml is also included as a negative (non-FtsZ-targeting)control. Experimental conditions for the polymerization studies were 5 �MFtsZ, 50 mM Tris-HCl (pH 7.4), 50 mM KCl, and 10 mM magnesium acetate.Polymerization reactions were initiated by the addition of 4 mM GTP at thetimes indicated by the arrows.

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Thus, unlike many clinical MRSA drugs, including vancomycin,daptomycin, and the streptogramins, TXY436 is orally bioavail-able.

TXY436 is orally efficacious in vivo against both MSSA andMRSA systemic infections. Armed with the pharmacokinetic re-sults described above, we next evaluated the oral in vivo efficacy ofTXY436 using a mouse peritonitis model of systemic infectionwith both MSSA and MRSA. In the MSSA studies, four groups ofsix female Swiss Webster mice were inoculated intraperitoneallywith a lethal inoculum of S. aureus ATCC 19636. The four groupsof infected mice were treated as follows: group 1, untreated; group2, treated p.o. with 10 mM citrate vehicle alone; group 3, treatedp.o. with TXY436 at 128 mg/kg administered in four divided dos-es; and group 4, treated p.o. with PC190723 at 128 mg/kg admin-istered as a suspension in four divided doses. All untreated micedied within 1 day of infection. In addition, none of the micetreated with citrate vehicle alone or with PC190723 survived be-yond 1 day postinfection (Fig. 9A). Thus, oral administration ofPC190723 itself was not efficacious in vivo. It is likely that the lackof oral efficacy associated with PC190723 is related to the com-pound being administered as a suspension (due to its poor solu-bility), which may, in turn, limit the oral bioavailability of thecompound. In striking contrast to PC190723, oral administrationof TXY436 proved to be highly efficacious, with 100% of theTXY436-treated mice surviving the infection.

In our MRSA studies, three groups of six mice were inoculatedintraperitoneally with a lethal inoculum of MRSA ATCC 43300.The three groups of infected mice were treated as follows: group 1,untreated; group 2, treated p.o. with 10 mM citrate vehicle alone;and group 3, treated p.o. with TXY436 at 192 mg/kg administeredin six divided doses. All untreated mice died within 2 days of

infection. Similarly, none of the mice treated with citrate vehiclealone survived beyond 2 days postinfection (Fig. 9B). However, inthe TXY436-treated group, 5 of 6 mice (83%) survived for 3 dayspostinfection, and 4 of 6 mice (67%) survived the infection en-

FIG 7 Fluorescence micrographs of B. subtilis FG347 bacteria that express a GFP-tagged, Z-ring marker protein, ZapA. The bacteria were cultured for 1 h in thepresence of DMSO (solvent control) (A), TXY436 at 3 �g/ml (B), or PC190723 at 3 �g/ml (C), at which point GFP-ZapA expression was induced by the additionof xylose. The bacteria were incubated for an additional hour at 37°C and then visualized by fluorescence microscopy. The arrows in panel A highlight FtsZZ-rings at midcell.

TABLE 4 Impact of mouse serum on the antistaphylococcal activities ofTXY436 and PC190723

Compound

S. aureus 8325-4 MIC (�g/ml)

No serum 50% mouse serum

TXY436 0.5 2.0PC190723 0.5 2.0Vancomycin 1.0 2.0

FIG 8 Time-dependent plasma concentrations of TXY436 (A) and PC190723(B) after a single i.v. administration of TXY436 at 5 mg/kg (Œ) or a single p.o.administration of TXY436 at 10 mg/kg (Œ) to male BALB/c mice.

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tirely. Thus, TXY436 is not only orally efficacious against systemicMSSA infection but also against systemic MRSA infection.

In both the MSSA and the MRSA studies, an additional positivecontrol group of six infected mice was also included. This groupwas treated i.v. with vancomycin at doses of 16 mg/kg in the MSSAstudies and 24 mg/kg in the MRSA studies. As anticipated, allvancomycin-treated mice survived their infections.

TXY436 is minimally toxic to mammalian cells. An impor-tant criterion for the potential clinical utility of an antibacterialagent is minimal toxicity against mammalian cells. In this connec-tion, we used a 3-day tetrazole (MTT)-based assay to assess the

cytotoxicity, if any, of TXY436 against Vero African green monkeykidney epithelial cells. TXY436 was found to be minimally toxic toVero cells, with a 50% inhibitory concentration (IC50) of 64 �g/ml. Significantly, this IC50 is 64� to 128� the antistaphylococcalMICs of TXY436 (which range from 0.5 to 1.0 �g/ml). Albeitderived from a single mammalian cell line, this differential sug-gests that TXY436 is associated with a significant therapeutic win-dow. Future studies with additional mammalian and human celllines will be directed toward assessing the general applicability ofthis therapeutic window.

In conclusion, TXY436 is a prodrug of the FtsZ-targeting benz-amide PC190273 with enhanced formulation properties as well asoral efficacy in vivo against both MSSA and MRSA infections.These characteristics, coupled with a minimal potential for toxic-ity to mammalian cells, make TXY436 a worthy candidate forfurther development as a clinically useful agent to treat staphylo-coccal infections.

ACKNOWLEDGMENTS

This study was supported by research agreements between TAXIS Phar-maceuticals, Inc., and both Rutgers Robert Wood Johnson MedicalSchool (D.S.P.) and Rutgers Ernest Mario School of Pharmacy (E.J.L.).

We are indebted to Nancy Connell (Rutgers New Jersey MedicalSchool, Newark, NJ) for assistance with the mammalian cytotoxicity stud-ies. We are also indebted to Glenn W. Kaatz (John D. Dingell VA MedicalCenter, Detroit, MI) for providing the S. aureus 8325-4 strain and toRichard Losick (Harvard University, Boston, MA) for providing the B.subtilis FG347 strain.

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TABLE 5 Pharmacokinetic parameters of the TXY436 prodrug and the PC190723 product following a single intravenous or peroral administrationof TXY436 to male BALB/c micea

Compound Route Dose (mg/kg) tmax (h) C0b (ng/liter) AUClast (ng/liter·h) t1/2 (h) CL (ml/min·kg) Vss (liters/kg) %Fc

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a Parameters were calculated by analysis of the plasma concentration versus time plots shown in Fig. 8 using the sparse sampling mode in the noncompartmental analysis (NCA)module of the Phoenix WinNonlin version 6.3 software package. NA, not applicable.; i.v., intravenous; p.o., peroral.b An extrapolated concentration was used for the i.v. group.c The AUClast value was used to calculate the %F value.

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